Project 3 ? Homologous Recombination Repair PROJECT SUMMARY/ABSTRACT By eliminating DNA double-strand breaks (DSBs) and other deleterious chromosomal lesions, such as those arising from replication fork (RF) stalling or collapse, homologous recombination-mediated DNA repair (HRR) is critical for the maintenance of genome stability. In humans, several tumor suppressors, including BRCA2 and PALB2, function to ensure the successful completion of HRR. As such, mutations in these proteins and their partners cause hypersensitivity to agents that damage DNA or replication forks and can lead to cancer formation. During the HRR process, a primary lesion is resected nucleolytically to yield ssDNA, which becomes occupied by the abundant single-stranded DNA binding protein RPA. For HRR to occur, RPA must be replaced by the RAD51 recombinase enzyme, which catalyzes the search for a DNA homolog to initiate the HRR reaction. RAD51 functions within the context of a filamentous intermediate on ssDNA, commonly referred to as the presynaptic filament. The handoff from RPA to RAD51 in presynaptic filament assembly is facilitated by HRR factors known as ?mediators?. We will capitalize on major advances made during SBDR-3 to dissect the mechanistic roles of the human HRR mediators. A combination of biochemical, biophysical, genetic, and cell- based approaches will be employed in four specific aims to define the action of the XPG-PALB2-BRCA2-DSS1 complex and of several conserved RAD51 paralogs in the assembly of the presynaptic filament. We will (1) decipher the presynaptic functions of BRCA2-DSS1 and PALB2, (2) determine the molecular mechanism of the role of XPG, (3) elucidate the structural biochemistry of the RAD51 paralogs, and (4) analyze the promotion of presynaptic filament assembly by these mediators at the single-molecule level. The results from our endeavors will allow us to formulate detailed models to elucidate HRR mechanisms in human cells. Given the importance of HRR in tumor suppression, our work has direct, strong relevance to cancer biology, and has the potential to identify strategies and targets for therapeutic intervention in cancer treatment. The success of our project studies and of the entire SBDR program is assured by the synergistic research structure that we have established and the exceptional services provided by the two research cores.